CN117503933A - Prevention and treatment of diabetic nephropathy - Google Patents

Abstract

Embodiments herein disclose methods related to diabetic nephropathy (DN); methods of preventing the onset of DN and preventing progression of DN, and treating DN in a diabetic subject comprising administering DRIN as an inhibitor of CXCL8 receptor CXCR1 and CXCR2 activation. Parixin and/or Ladarixin.

Description

Prevention and treatment of diabetic nephropathy

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese patent application No. 201780061270.5. According to 35 U.S.C. §119(e), this application claims the benefit of U.S. Provisional Application No. 62/403,368 filed on October 3, 2016, which is incorporated herein by reference in its entirety.

Field of the Invention

Embodiments disclosed herein relate to diabetic nephropathy (DN); in particular, the embodiments relate to methods of preventing and treating DN in diabetic subjects.

Background of the Invention

Diabetic nephropathy (DN) is the most common cause of renal failure and accounts for approximately half of patients receiving long-term renal dialysis and end-stage renal disease. It is one of the most serious complications faced by diabetic patients, with an incidence of approximately 40% in this patient population. Certain risk factors significantly increase the likelihood of developing this condition in diabetic patients. These include poor control of blood sugar levels, length of time with diabetes, being overweight, and the presence of high blood pressure (over 130/80 mm Hg).

Standard tests used to assess kidney function are measurement of the glomerular filtration rate (GFR), the rate of flow of filtered fluid through the kidneys, and screening for proteinuria in the urine.

The glomeruli constitute the filtration system of the kidneys, which allows for the selective ultrafiltration of plasma into the urine. This barrier is freely permeable to water, small and medium-sized solutes, including proteins with a molecular weight lower than albumin, but prevents larger molecules and proteins from entering the urine. Any damage to the glomeruli will affect the kidneys' ability to control the passage of substances from the blood into the urine.

The filtration apparatus of the glomerulus is composed of three layers: the fenestrated endothelium, the glomerular basement membrane (GBM), and the epithelial podocytes. The function of the glomerular filtration barrier depends on the integrity and functionality of all three layers.

In the urine of a normal healthy kidney, less than 0.05% of plasma albumin is present. This small amount of albumin is filtered into the urine at the glomerular level and subsequently absorbed and degraded by the proximal tubular cells. The remaining fragments are reabsorbed into the tubular lumen as albumin fragments.

Many pathophysiological elements associated with diabetes induce glomerular damage and, therefore, damage to the filtration system, leading to excretion of protein into the urine, particularly albuminuria. Thus, elevated levels of protein in the urine are the first sign of kidney damage due to diabetes. The presence of protein in the urine then causes tubular damage and nephron loss.

DN is divided into two main stages, depending on how much albumin is lost through the kidneys: microalbuminuria and macroalbuminuria. Microalbuminuria is characterized by an amount of albumin flowing into the urine of 30 to 300 mg per day. It is sometimes called incipient nephropathy. Macroalbuminuria is characterized by an amount of albumin flowing greater than 300 mg per day.

Microalbuminuria is usually the first sign that DN has developed. However, it is not necessarily associated with progression to macroalbuminuria and loss of renal function. In most cases, microalbuminuria can return to normoalbuminuria, but it may also continue or progress to macroalbuminuria at roughly the same level. On the contrary, once macroalbuminuria develops, the condition is irreversible and causes the glomerular filtration rate (GFR) to drop to end-stage renal failure. Once the patient is at this stage of the disease, irreversible damage will occur to the structure of the glomerulus.

The destruction of the glomerular filtration barrier is associated with many histopathological changes in the glomerulus. The structural abnormalities of diabetic nephropathy in patients with type 1 and type 2 diabetes are similar. The earliest morphological change in DN is mesangial expansion, which is due to an increase in the number of mesangial cells and an increase in the synthesis of mesangial matrix and a decrease in its degradation. Podocytes and endothelial cells appear to play a role in this process by stimulating the mesangial cells to respond by increasing mesangial matrix deposition. As the disease progresses, mesangial matrix and cells continue to accumulate, leading to thickening of the glomerular basement membrane and the development of many sclerotic nodules (nodular transformation), ultimately leading to glomerular sclerosis. When these structural lesions lead to functional impairment, they are in a very advanced stage, and current treatments can slow down but not prevent progression to end-stage renal disease (ESRD) (Mauer et al., J Clin Invest 1984, 74: 1143; Lewis et al., N Engl J Med 1993, 329: 1456).

Therefore, it is important to identify therapeutic strategies that can intervene in the early causes of glomerular structural and functional destruction before irreversible damage to the glomerular filtration system occurs and overt signs or symptoms of renal disease (such as macroalbuminuria and/or reduced GFR) occur.

Current treatments for diabetic nephropathy aim to prevent or delay progression to renal failure, primarily by reducing factors known to significantly increase the likelihood that diabetic patients will develop the pathology, such as cardiovascular disease, poor control of blood glucose levels, and hypertension. However, despite overall improvements in treatments over the past few years, these have proven to be limited in effectiveness, as evidenced by the increase in the number of patients who ultimately develop diabetic nephropathy. Recent research has focused on the development of potential new therapies that target pathways thought to promote the progression of kidney disease, such as inhibitors of advanced glycation end products (AGEs), protein kinase C, vitamin D, or endothelin 1. However, these potential alternative therapies have not yet been successfully translated into clinical practice.

Interleukin-8 (IL8; CXCL8) is considered to be a major mediator of PMN (polymorphonuclear neutrophil) recruitment and is involved in a variety of pathologies, including psoriasis, rheumatoid arthritis, chronic obstructive pulmonary disease, and ischemia/reperfusion injury in transplanted organs (Griffin et al., Arch Dermatol 1988, 124:216; Fincham et al., J Immunol 1988, 140:4294; Takematsu et al., Arch Dermatol 1993, 129:74; Liu et al., 1997, 100:1256; Jeffery, Thorax 1998, 53:129; Pesci et al., Eur Respir J. 1998, 12:380; Lafer et al., Br J Pharmacol. 1991, 103:1153; Romson et al., Circulation 1993, 67: 1016; Welbourn et al., Br J Surg. 1991, 78: 651; Sekido et al., Nature 1993, 365, 654). The biological activity of IL8 is mediated by interaction with two receptors belonging to the 7TM-GPCR family, CXCR1 and CXCR2, expressed on the surface of human PMNs.

A number of studies have demonstrated that urinary IL8 levels are increased in patients with microalbuminuria, suggesting that IL8 may be used in combination with at least one other marker such as IP-10, IL-6, MIP-1δ or MCP1 in a prognostic approach to identify a population of microalbuminuric patients who may experience progressive renal function decline (Tashiro et al., J Clin Lab Anal 16:1-4, 2002).

Reparixin and Ladarixin are noncompetitive allosteric inhibitors of CXCR1 and CXCR2, the cognate receptor of IL8 (CXCL8), which can block a series of activities related to IL8 signal transduction, including leukocyte recruitment and other inflammatory responses, without affecting the binding between ligand and receptor (Bertini, R et al., Proc Nat Acad Sci USA 2004, 101: 11791).

Reparixin is the INN name of R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (formerly known as Repertaxin or DF 1681Y), and it was first disclosed in International Application WO0024710. Further description of Reparixin and its use in preventing diabetes is found in U.S. Patent Application Publication No.: US2015/0011639. The use of Reparixin in reducing or inhibiting graft rejection in individuals receiving islet cell transplants is described in U.S. Patent Application Publication No.: US2012/0202884. However, the preventive and therapeutic effects of Reparixin on diabetic nephropathy have not been proposed or explored. The use of Reparixin in cancer treatment is described in WO2010/056753. The contents of each of these patents are incorporated herein by reference in their entirety.

Ladarixin is the INN name for R(-)-2-[(4′-trifluoromethanesulfonyloxy)phenyl]propionyl-methanesulfonamide sodium salt (formerly known as Meraxin or DF2156A). Ladarixin has been shown to inhibit CXCR1 and CXCR2 and is able to block and reverse type 1 diabetes in mice (Citro A. et al., Diabetes 2015, 64: 1329).

(2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propanoic acid (also known as DF2755Y) and its sodium salt (also known as DF2755A) are potent and selective dual inhibitors of CXCR1- and CXCR2-triggered PMN activity, which were first disclosed in WO2010/031835 and their use in the treatment of IL8-dependent pathologies such as transient cerebral ischemia, bullous pemphigoid, rheumatoid arthritis, idiopathic fibrosis, glomerulonephritis, and ischemia/reperfusion-induced injury.

The present inventors have now demonstrated the therapeutic efficacy of IL8 inhibitors in preventing proteinuria under diabetic conditions and protecting the kidney from the onset and progression of diabetic nephropathy (DN). IL8 has also been demonstrated to be involved in the early histopathological changes leading to the onset of kidney disease.

SUMMARY OF THE INVENTION

Specifically, embodiments of the present disclosure are based on experimental demonstration of the therapeutic efficacy of Reparixin in preventing and/or restoring mesangial expansion and podocyte damage in diabetic conditions and preventing further development and progression of diabetic nephropathy (DN).

Therefore, the first object of the present disclosure is to provide a method for treating diabetic nephropathy or preventing, reducing the risk or delaying the onset or progression of diabetic nephropathy, which comprises administering to a subject in need thereof an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

The second object of the present disclosure is to provide an IL8 inhibitor for the treatment of diabetic nephropathy in a subject or the prevention, risk reduction or delay of the onset or progression of diabetic nephropathy, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

The third object of the present disclosure is to provide the use of an IL8 inhibitor in the preparation of a medicament for the treatment of diabetic nephropathy in a subject or the prevention, risk reduction or delay of the onset or progression of diabetic nephropathy, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

The fourth object of the present disclosure is to provide an IL8 inhibitor for the treatment of diabetic nephropathy in a subject or the prevention, risk reduction or delay of the onset or progression of diabetic nephropathy, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as 1adarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y)) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

A fifth object of the present disclosure is to provide a method of treatment, comprising determining the level of IL8 in a urine sample from a subject, and administering an effective amount of Reparixin and/or Ladarixin to the subject when the IL8 level is at least 3 times higher than a reference level.

A sixth object of the present disclosure is to provide a method for treating hyperglycemia, comprising diagnosing a subject with hyperglycemia, and administering an effective amount of Reparixin and/or Ladarixin to the subject.

A seventh object of the present invention is to provide a method for treating hyperglycemia, which comprises administering an effective amount of Reparixin and/or Ladarixin to a patient in need thereof.

An eighth object of the present disclosure is to provide a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for use in the treatment of diabetic nephropathy in a subject or the prevention, risk reduction, or delay of the onset or progression of diabetic nephropathy. In one embodiment, a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin is administered to treat diabetic nephropathy in a subject or to prevent or reduce the risk of diabetic nephropathy or delay the onset or progression of diabetic nephropathy, wherein the subject has an IL8 level above 2.4 pg/ml. In one embodiment, a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin is administered for the treatment of diabetic nephropathy or the prevention, risk reduction, or delay of the onset or progression of diabetic nephropathy in a subject, wherein the subject's IL8 level is 3-fold higher than a reference level.

A ninth object of the present invention is to provide a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for the preparation of a medicament for the treatment of diabetic nephropathy or the prevention, risk reduction, or delay of the onset or progression of diabetic nephropathy in a subject.

A tenth object of the present disclosure is to provide use of a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for the treatment of diabetic nephropathy or the prevention, risk reduction, or delay of the onset or progression of diabetic nephropathy in a subject.

An eleventh object of the present disclosure is to provide use of a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for the preparation of a medicament for the treatment of diabetic nephropathy or the prevention, risk reduction, or delay of the onset or progression of diabetic nephropathy in a subject.

Therefore, in one embodiment of the above-mentioned objects of the present invention, in the method or use, the subject has been diagnosed with diabetes.

Therefore, in one embodiment of the above-mentioned various objects of the present invention, in the method or use, the subject has been diagnosed with type 1 diabetes.

Therefore, in one embodiment of each of the above objects of the present invention, in the method or use, the subject has been diagnosed with type 2 diabetes.

According to another embodiment of the present invention according to each of the above objects, in combination with any of the preceding embodiments, in the method or use, the subject has microalbuminuria.

According to another embodiment of the above-mentioned various objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the urine IL8 level of the subject is higher than an IL8 reference standard. Preferably, the reference standard is the urine IL8 level of a healthy individual who does not suffer from any kidney disease.

According to another embodiment of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject's urine IL8 level is above 2.41 pg/ml.

According to another embodiment of the above objects of the invention, in combination with any of the preceding embodiments, in said method or use, the subject has a GFR (glomerular filtration rate) value higher than 60 ml/min/1.73 m ² , preferably higher than 90 ml/min/1.73 m ² .

According to another embodiment of the above-mentioned various objects of the present invention, in combination with any of the preceding embodiments, the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring the level of IL8 in a sample obtained from a subject; (b) comparing the measured level of IL8 to an IL8 reference; (c) when the measured level of IL8 is higher than the IL8 reference, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably a compound selected from the group consisting of: R(-)-2-[(4-isobutyl)- [(4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propanoic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A), wherein the IL8 reference is the average urine IL8 level of a statistically significant number of non-diabetic individuals who do not suffer from any kidney disease and do not suffer from inflammatory diseases.

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring the level of IL8 in a sample obtained from the subject; (b) when the measured IL8 level is above 2.4 lpg/ml, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably selected from the following Compounds: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or its salt, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or its salt, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A).

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) determining whether the subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514; (b) when the subject has at least one of the SNPs, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CX CR2 inhibitors, more preferably CXCR1 and CXCR2 inhibitors, even more preferably compounds selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as Reparixin) or its salt, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as 1adarixin) or its salt, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A).

In one embodiment of the first aspect of the present invention, in combination with any of the preceding embodiments, the method further comprises measuring protein levels in a urine sample from the subject.

In one embodiment of the first aspect of the present invention, in combination with any of the preceding embodiments, the method further comprises selecting a subject having microalbuminuria.

In one embodiment of the first aspect of the present invention, in combination with any of the preceding embodiments, the method further comprises obtaining a urine sample from the subject for analysis of urine protein levels.

In one embodiment of the first aspect of the present invention, in combination with any of the preceding embodiments, the method further comprises comparing the measured protein level to a urine protein reference.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, the first reference is the protein level in a urine sample obtained from a normal healthy subject not suffering from any kidney disease.

In one embodiment of any of the above methods or uses, the subject has normal proteinuria or increased proteinuria.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, the method further comprises measuring the level of IL8 in a sample obtained from the subject.

In one embodiment of any of the above methods or uses, the sample is a urine, blood, serum or plasma sample.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, the method further comprises comparing the measured IL8 level with an IL8 reference level.

In one embodiment of any of the above methods or uses, the reference level of IL8 is the level of IL8 in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

In one embodiment of any of the above methods or uses, the reference level of IL8 is the level of IL8 in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease or any inflammatory condition.

In one embodiment of the first aspect of the invention, in combination with any of the preceding embodiments, the method further comprises selecting a subject wherein the level of IL8 measured in the urine is above 2.41 pg/ml.

In one embodiment of the first object of the invention, in combination with any of the preceding embodiments, the method further comprises determining whether the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s1 3006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows experimental data showing urinary albumin excretion levels (μg) measured over 24 hours in control mice (CTRL) and mice treated with Reparixin (REPA) as described in Example 2.

FIG. 2A shows experimental data showing the amount of actin expression measured by phalloidin staining in control (CTRL), IL8-treated (IL8), and IL8- and reparixin-treated (IL8reparixin) podocytes.

Figure 2B shows experimental data showing synaptopodin mRNA expression compared to GAPDH mRNA expression measured in control (CTRL), IL8-treated (IL8), and IL8 and reparixin-treated (IL8reparixin) podocytes.

FIG. 3A shows experimental data quantifying IL8 expression in patients with Mesangial Expansion (Mes Exp), Nodular Transf, or Sclerosis, expressed as a percentage of glomerular area.

FIG3B shows experimental data showing IL8 expression levels in glomeruli of control individuals and patients with type 2 diabetes and DN measured by quantitative PCR, expressed as fold increase relative to control.

FIG4 shows experimental data showing mean urinary IL8 levels measured in T2D normoalbuminuric (Normo), microalbuminuric (Micro), macroalbuminuric (Macro), or control (CTRL) patients.

FIG5A shows experimental data showing mean ACR values in patients, wherein the expression of IL8 corresponds to the first (Q1), second (Q2), third (Q3), or fourth (Q4) quartile;

FIG5B shows experimental data showing ACR values in patients with IL8 expression below (Q1-Q2) or above (Q3-Q4) the median.

FIG6 shows a model showing the mechanism by which Reparecin acts to prevent cytoskeletal remodeling.

The experimental data shown in FIG. 7A and FIG. 7B show that the expression of IL8 has a peak at the early injury stage and gradually decreases after the loss of cellularity of the renal parenchyma and the onset of fibrosis.

Figure 7C shows experimental data showing fold increase in IL8 mRNA expression in diabetic samples compared to controls. mRNA levels were analyzed by RT-PCR.

Figure 7D shows experimental data showing fold increase in CXCR-1 mRNA expression in diabetic samples compared to controls. mRNA levels were analyzed by RT-PCR.

Figure 7E shows experimental data showing fold increase in CXCR-2 mRNA expression in diabetic samples compared to controls. mRNA levels were analyzed by RT-PCR.

FIG8A shows experimental data showing that patients with microalbuminuria exhibited higher urinary IL8 levels compared to normoalbuminuric patients.

The experimental data shown in FIG8B show that for all 389 patients and their normoalbuminuric and microalbuminuric subsets, those patients who tested positive for IL8 in urine also presented significantly higher ACR values.

The experimental data shown in FIG8C show that for all 389 patients and their normoalbuminuric and microalbuminuric subsets, those patients with a positive urine IL8 test had significantly greater GFR slopes in both the microalbuminuric and normoalbuminuric groups.

The experimental data shown in Figure 8D show that for all 389 patients and their normoalbuminuric and microalbuminuric subsets, those patients above the median distribution of IL8 in both the normoalbuminuric and microalbuminuric groups showed significantly higher ACR than those patients below the median.

FIG8E shows experimental data showing event risk for all 389 patients and their normoalbuminuric and microalbuminuric subsets.

FIG8F shows a graph showing IL8 expression in normoalbuminuria and microalbuminuria subsets.

FIG. 9A shows experimental data depicting cells expressing IL8 under normoglycemia and hyperglycemia for 14 days.

FIG. 9B shows experimental data depicting cells expressing CXCR-1 under normoglycemia and hyperglycemia for 14 days.

FIG. 9C shows experimental data of cells expressing CXCR-2 under normoglycemia and hyperglycemia for 14 days.

FIG. 10A shows experimental data showing the mean cell area of podocytes with or without reparixin treatment under the indicated conditions.

FIG. 10B shows experimental data showing mean cellular fluorescence intensity of podocytes with or without reparixin treatment under the indicated conditions.

FIG. 11A shows experimental data showing in vivo blood glucose levels at the indicated time points in control or REPA-treated DN db/db diabetic mice.

FIG. 11B shows experimental data showing in vivo UAE levels at the indicated time points in control or REPA-treated DN db/db diabetic mice.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein, and thus may be varied. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the claims.

Definitions of commonly used terms in molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3) or the 2015 digital online version at merckmanuals.com; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Werner Luttmann’Immunology, published by Elsevier, 2006; Janeway’Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (editors), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin’Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-14 49659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JOhn Wiley and Sons, 2014 (ISBN 0471503 38X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (editor), JOhn Wiley and Sohs, Inc., 2005; Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entirety; furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Unless otherwise indicated, the present invention is performed using standard procedures known to those skilled in the art, such as Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel et al., ed., John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI) (John E. Coligan et al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al., ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998), Methods in Molecular biology, Vol. 180, Transgenesis Techniques by Alan R. Clark editor, 2nd edition, 2002, Humana Press, and Marten H. Hofker and Jan van Deursen editors, Methods in Meolcular Biology, Vo. 203, 2003, standard procedures in Transgenic Mouse, all of which are incorporated herein by reference in their entirety.

It should be understood that the present invention is not limited to the particular methodology, protocols, reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the claims.

Except in the operating examples, or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about." When used in conjunction with a percentage, the term "about" shall mean ±1%.

For the purpose of description and disclosure, all patents and publications identified are explicitly introduced into this paper by reference, and the methods described in these publications, for example, can be used in combination with the present invention. These publications are provided only because they are disclosed before the filing date of this application. Any content in this regard should not be interpreted as admitting that the inventor has no right to precede such disclosure due to prior invention or any other reason. All statements about dates or statements about the contents of these documents are based on information available to the applicant and do not constitute any recognition of the correctness of the dates or contents of these documents. The terms "object", "individual", "patient" and "human" used herein are used interchangeably to represent mammals, such as dogs, cats, cattle and horses, and preferably humans.

In one embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly humans. Specifically, it refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of wetting agent or emulsifier or pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated into suppositories using traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th edition, edited by Gennaro (Mack Publishing Co., 1990). The formulation should be suitable for the mode of administration.

As used herein, the term "comprising" is used to refer to the methods and their corresponding components that are essential to the present invention, but are open ended to include unspecified elements, whether essential or non-essential. The use of "comprising" means inclusion rather than limitation.

As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel features or functional characteristics of the embodiments of the invention.

The term "consisting of" refers to the compositions, methods, and respective components thereof as described herein, excluding any elements not recited in the description of the embodiment.

The terms "diabetic kidney disease," "DKD," "diabetic nephropathy," and "DN" are used interchangeably herein to refer to any loss of kidney structural integrity and function resulting in the leakage of certain nutrients, such as protein, into the urine rather than being reabsorbed back into the blood.

The terms "disease" or "condition" are used interchangeably herein to refer to any alternation in the state of the body or some organ that interrupts or disrupts functional performance and/or causes symptoms such as discomfort, dysfunction, pain, or even death in the patient or those in contact with the patient. Disease or condition may also refer to distemper, ailment, ailment, malady, disorder, sickness, illness, complaint, or affectation.

When used in the context of therapeutic or prophylactic treatment, the term "in need thereof" means having a disease, being diagnosed with a disease, or in need of prevention of a disease, such as for a subject at risk of developing a disease. Thus, a subject in need thereof may be a subject in need of treatment or prevention of a disease.

As used herein, in one embodiment, the phrase "preventing the onset of diabetic nephropathy" means stopping, hindering and/or slowing the initial occurrence of more than 30 mg of albumin in the urine of a diabetic subject or a subject with a diabetic condition.

In another embodiment, the terms "prevent/prevention" as used herein in the context of the onset of DN and the progression of DN in diabetic subjects or subjects with a diabetic condition refer to stopping, hindering, and/or slowing the onset of developing adverse reactions and symptoms associated with medical conditions associated with DN, such as loss of kidney structural integrity and function, resulting in leakage of certain nutrients into the urine instead of being reabsorbed back into the blood, such as leakage of protein into the urine.

As used herein, the phrase "preventing the progression of diabetic nephropathy" means stopping, arresting and/or slowing the sustained occurrence/recurrence of more than 30 mg albumin/day in the urine of a diabetic subject or a subject with a diabetic condition.

As used herein, in one embodiment, the term "treatment (treat/treatment/treating)" or "improvement" refers to therapeutic treatment, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progress or severity of DN and renal failure. If one or more symptoms or clinical markers are reduced, treatment is usually "effective". For example, in DN, "effective treatment" refers to the protein excreted into the urine is reduced to the normal protein/albumin range and maintained within the normal range for at least one week of treatment. In one embodiment, treatment as described herein can reduce proteinuria and maintain the normal range of protein/albumin for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks or longer, for example, at least 20 weeks (or 5 months), 6 months or longer. In another embodiment, if the progression of the disease slows down or stops, treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, such as IL8 or other markers disclosed in U.S. Patent Application Publication No.: US 2006/0240437, but also the cessation or at least slowing of symptom progression or worsening compared to the expected symptoms in the absence of treatment. Beneficial or desired clinical results include, but are not limited to: alleviating one or more symptoms, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, delaying or slowing the progression of the disease, improving or alleviating the disease state, alleviating (whether partially or completely) and/or reducing mortality. For example, if the condition is stable or the urine protein/albumin level is normalized, the treatment is considered to be effective. The term "treatment" of a disease also includes situations in which the symptoms or side effects of the disease (including palliative care) are alleviated.

As used herein, the term "inhibitor" as used herein refers to any synthetic/natural organic or organic/inorganic molecule that antagonizes the naturally occurring signal transduction events triggered by the binding activation of IL8 chemokine to its receptor or CXCR1 and/or CXCR2.

The term "administering" as used herein refers to placing the IL8 inhibitor disclosed herein in an object by a method or approach that results in at least partial delivery of an agent/drug at a desired site. A pharmaceutical composition comprising a compound disclosed herein can be administered by any suitable approach that results in effective treatment in an object. "Administering" refers to oral administration ("po") to an object, administration as a suppository, topical contact, intravenous ("iv"), intraperitoneal ("ip"), intramuscular ("im"), intralesional, intranasal or subcutaneous ("sc") administration or implantation of a sustained-release device, such as a miniature osmotic pump. Administered by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal or transdermal) administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, ventricular and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposome preparations, intravenous infusions, transdermal patches, etc.

The term "systemic administration/systemic administration" refers to the administration of a compound or composition to a mammal so that the compound or composition is delivered to a site in the body via the circulatory system, including methods of targeting the site of drug action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (i.e., except through the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration, provided that systemic administration as used herein does not include administration directly to a brain region by means other than through the circulatory system, such as intrathecal injection and intracranial administration.

As used herein, in one embodiment, the term "increased proteinuria" means an increase of at least 10% in the protein level in a urine sample compared to a urine protein reference level. In another embodiment, "increased proteinuria" means urine excretion greater than 30 mg albumin/24 hr day.

As used herein, the terms "increased" or "increase" or "elevation" generally mean an increase in a statistically significant amount; for the avoidance of doubt, the terms "increase" or "enhancement" mean an increase of at least 10% compared to a reference level, such as an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

The terms "decrease/reduce/reduction", "lower/lowering" or "inhibit" are generally used herein to refer to a statistically significant amount of reduction. For example, "reduce" or "inhibit" means a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% reduction (e.g., no level or undetectable level compared to a reference level), or any reduction between 10-100% compared to a reference level. In the context of markers or symptoms, these terms mean a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to a level accepted within the normal range for individuals not suffering from a given disease.

The term "statistically significant" or "significant" refers to statistical significance, and generally means a difference of two standard deviations (2SD) or greater.

The term "normalization" refers to a change in urine protein/albumin levels from elevated urine protein/albumin levels to within the normal range. "Normalization" refers not only to activities that promote a decrease in abnormally high urine protein/albumin levels, but also to the maintenance of such levels over a long period of time, e.g., at least one week for administration of a single unit dose of a pharmaceutical composition as described herein.

The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variants thereof, as used herein, are used interchangeably when they refer to compositions, carriers, diluents and agents and indicate that the material can be administered to a mammal or administered to a mammal without producing undesirable physiological effects, such as nausea, dizziness, stomach discomfort, etc. Unless so desired, a pharmaceutically acceptable carrier will not promote the generation of an immune response to the medicament mixed therewith. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well known in the art and does not need to be limited based on the formulation. Such compositions are usually prepared as injectable liquid solutions or suspensions, but can also be prepared in a solid form suitable for dissolution or suspension in a liquid before use. The formulation can also be emulsified or present as a liposomal composition. The active ingredient can be mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient and in an amount suitable for use in the methods of treatment described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, and the like and combinations thereof. In addition, if necessary, the composition may contain a small amount of auxiliary substances that enhance the effectiveness of the active ingredient, such as wetting agents or emulsifiers, pH buffers, etc. The therapeutic composition of the present invention may contain pharmaceutically acceptable salts of its components. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids such as acetic acid, tartaric acid, mandelic acid (formed with the free amino groups of the polypeptide). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any substance except the active ingredient and water, or contain buffers, such as sodium phosphate, saline or both at physiological pH values, such as phosphate buffered saline. In addition, aqueous carriers can contain a variety of buffer salts and salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. In addition to water, the liquid composition may also include a liquid phase. Examples of such additional liquid phases are glycerol, vegetable oils (such as cottonseed oil) and water-oil emulsions. The amount of the active agent effective for treating a specific disorder or condition in the methods described herein will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A.Osol, a standard reference in this technical field. For example, a parenteral composition suitable for injection is prepared by dissolving 1.5% by weight of the active ingredient in 0.9% sodium chloride solution.

In one embodiment, a "pharmaceutically acceptable" carrier does not include in vitro cell culture medium.

The terms "sustained release" and "extended release" are used in their conventional sense to refer to a drug formulation that provides gradual release of the drug over an extended period of time, e.g., 12 hours or more, and preferably (although not necessarily) results in a substantially steady state blood level of the drug over the extended period of time.

As used herein, the term "delayed release" refers to a drug formulation that passes through the stomach intact and dissolves in the small intestine.

The terms "controlled release", "sustained release", "extended release" and "timed release" are intended to refer interchangeably to any drug-containing formulation in which the drug is not immediately released, i.e., with a "controlled release" formulation, oral administration does not result in immediate release of the drug into the absorption pool. The term may be used interchangeably with "non-immediate release" as defined in Remington: The Science and Practice of Pharmacy, 21st edition, Lippencott Williams & Wilkins (2006).

As used herein, the term "subtherapeutic dose" refers to a dose of a pharmacologically active agent: the dose of the pharmacologically active agent administered or the actual level of the pharmacologically active agent in a subject that is functionally insufficient to induce the desired pharmacological effect in the subject (e.g., to obtain analgesic, anticonvulsant, antidepressant, anti-inflammatory, antihypertensive, cardioprotective or organ protective effects) or is quantitatively less than the established therapeutic dose of the specific pharmacological agent (e.g., disclosed in references consulted by those skilled in the art, such as Physicians' Desk Reference, 62nd Edition, 2008, Thomson Healthcare or Brunton et al.; Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition, 2006, McGraw-Hill Professional). "Subtherapeutic dose" can be defined relatively (i.e., as a percentage amount (less than 100%) of the amount of the pharmacologically active agent conventionally administered). For example, a subtherapeutic dose can be about 1% to about 75% of the amount of the pharmacologically active agent conventionally administered. In some embodiments, a subtherapeutic dose may be about 75%, 50%, 30%, 25%, 20%, 10% or less of the amount of the pharmacologically active agent conventionally administered.

As used herein, in the context of administering an IL8 inhibitor, the term "therapeutically effective amount" means an amount or dose of an IL8 inhibitor that, when administered to a diabetic individual exhibiting diabetic nephropathy or an individual with a diabetic condition, is therapeutically effective enough to reduce the development of one or more symptoms of the disease, condition or disorder being treated (e.g., an amount sufficient to reduce the level of protein in the urine of a diabetic individual exhibiting DN).

As used herein, in the context of administering an IL8 inhibitor, the term "prophylactically effective amount" means the amount or dosage of an IL8 inhibitor when administered to a diabetic individual who does not suffer from diabetic nephropathy or an individual with a diabetic condition, and refers to an amount of the drug sufficient to prevent the onset of DN symptoms or reduce the risk of the onset of DN symptoms (e.g., an amount sufficient to prevent an increase in protein levels in the urine of a diabetic individual), i.e., the biological or medical event that is sought to be prevented. In many cases, the prophylactically effective amount is the same as the therapeutically effective amount.

As used herein, the term "nephropathy" means kidney (renal) disease), also known as nephropathy, in which there is damage or disease to the kidneys.

As used herein, the term "diabetic condition" refers to a condition characterized by impaired glucose production and/or utilization, and includes diabetes (e.g., type 1 diabetes, type 2 diabetes, and gestational diabetes), prediabetes, metabolic syndrome, hyperglycemia, impaired glucose tolerance, and impaired fasting glucose.

As used herein, the term "diabetes" refers to a disease or condition generally characterized by metabolic defects in the production and utilization of glucose, which results in the inability to maintain adequate blood glucose levels in the body. A subject is identified as having diabetes if the subject has a fasting blood glucose level greater than 125 mg/dl, a glucose reading greater than 200 mg/dl after a 2-hour load, or an HbA1c level greater than or equal to 6.5%.

As used herein, the term "prediabetes" refers to a condition that is typically characterized by impaired glucose tolerance and typically precedes the onset of diabetes in a subject.

A subject was identified as having prediabetes if their fasting blood glucose level was greater than 100 mg/dl but less than or equal to 125 mg/dl, their 2-hour post-load glucose reading was greater than 140 mg/dl but less than 200 mg/dl, or their HbA1c level was greater than or equal to 6.0% but less than 6.5%.

As used herein, the term "hyperglycemia" refers to elevated blood glucose levels in the body that are caused by metabolic defects in the production and utilization of glucose. A subject is identified as hyperglycemic if their fasting blood glucose levels consistently exceed 126 mg/dl.

As used herein, the term "overweight" refers to an individual having a body mass index of 25 kg/m ² or more but less than 30 kg/m ² .

As used herein, the term "body mass index" or "BMI" refers to a measure of body mass to height ratio that estimates whether an individual's weight is appropriate for their height. As used herein, an individual's body mass index is calculated as follows: BMI = (pounds x 700) / (height in inches) ² or BMI = (kilograms) / (height in meters) ²

As used herein, the term "baseline body weight" refers to a subject's body weight at the start of treatment.

As used herein, the terms "obese" and "obesity" refer to individuals with a body mass index (BMI) of 30 kg/ ^m2 or higher due to excess adipose tissue. Obesity can also be defined based on body fat content: greater than 25% for men or greater than 30% for women. "Morbidly obese" individuals have a BMI greater than 35 kg/ ^m2 .

Embodiments of the present disclosure are based on experimental demonstration of the therapeutic efficacy of reparixin and/or ladarixin in preventing the onset and development and progression of diabetic nephropathy (DN).

In detail, the inventors have found that IL8 has a specific and reproducible expression pattern in the kidney in terms of the location and time of expression in diabetic animal models and diabetic patients. In fact, it is obvious from the experimental part that IL8 is found to be specifically expressed in endothelial cells and podocytes, and is expressed at a significant level only during mesangial expansion. On the contrary, during the subsequent progression of glomerular damage to nodular transformation and glomerulosclerosis, a consistent decrease in IL8 expression levels was observed. In diabetic animal models, the inventors demonstrated that blocking the KC/CXCR2 axis with Reparixin prevents increased urinary albumin excretion and mesangial expansion. In addition, IL8 shows direct damage to podocytes, which can be inhibited by administering CXCR1/2 inhibitors such as Reparixin. The experimental part shows that IL8 plays an important role in the initial pathological stage of the onset and development of diabetic nephropathy, stimulating mesangial expansion and podocyte damage. After this stage, its expression and pathogenic effects gradually decline. The inventors also identified urine IL8 levels corresponding to patients with the strongest response to anti-IL8 therapy, preferably patients with microalbuminuria.

Therefore, a first object of the present invention is a method for the treatment of diabetic nephropathy or the prevention, risk reduction or delay of the onset or progression of diabetic nephropathy, comprising administering to a subject in need thereof.

The second object of the present disclosure is to provide the use of an IL8 inhibitor in treating diabetic nephropathy or preventing, reducing the risk or delaying the onset or progression of diabetic nephropathy in a subject, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

The third object of the present invention is to provide the use of an IL8 inhibitor in the preparation of a medicament for treating diabetic nephropathy in a subject or preventing, reducing the risk or delaying the onset or progression of diabetic nephropathy. The IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

The fourth object of the present disclosure is to provide an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), which is used for the treatment of diabetic nephropathy in a subject or the prevention, risk reduction or delay of the onset or progression of diabetic nephropathy.

In one embodiment of the above objects of the present invention, in the method or use, the subject has been diagnosed with diabetes.

In one embodiment of the above objects of the present invention, in the method or use, the subject has been diagnosed with type 1 diabetes.

In one embodiment of the above objects of the present invention, in the method or use, the subject has been diagnosed with type 2 diabetes.

Thus, for example, in real life, it is expected that when an individual is diagnosed with diabetes (type 1 or type 2), an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably its sodium salt (hereinafter referred to as DF2755A), is immediately administered prophylactically to prevent the onset of diabetic nephropathy, which may develop with the persistence and management/treatment of the diabetic condition.

In one embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, the subject has normal proteinuria. In one embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, the subject has increased proteinuria. In one embodiment, the increased proteinuria is the excretion of at least 300 mg of albumin in 24 hours. In one embodiment, the protein measured is albumin.

In one embodiment of the above objects of the invention, in combination with any of the preceding embodiments, the subject suffers from microalbuminuria.

In one embodiment, the term "microalbuminuria" refers to any disease, disorder, condition, or health state in which urinary albumin is excreted in the urine at a rate of about 20-200 μg/minute or about 30-300 mg/24 hours (see, e.g., Abbott, KC et al., Arch. Internal Med. 754: 146-153 (1994), the teachings of which are incorporated herein by reference in their entirety). In another embodiment, the term "microalbuminuria" refers to any disease, disorder, condition, or health state in which albumin is excreted in the urine at 30 to 300 mg per day.

Methods for detecting and diagnosing microalbuminuria are well known to those skilled in the art and include radioimmunoassay, immunoassay with latex, fluorescence immunoassay, enzyme immunoassay, agglutination inhibition, immunoturbidimetric assay, immunoturbidimetric assay and radial immunodiffusion assay. For example, US5326707; US5492834, US5385847; US5750405, US20030027216; US20030232396, the contents of each of which are incorporated herein by reference in their entirety.

Thus, for example, when a diabetic individual is diagnosed with early diabetic nephropathy (as evidenced by the presence of microalbuminuria), an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a lysine salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), is immediately administered prophylactically to treat, prevent and/or delay the progression of diabetic nephropathy in diabetic individuals.

In another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the IL8 level in the subject's biological sample is higher than an IL8 reference standard, wherein the reference standard is the average IL8 level in corresponding biological samples of a statistically significant number of non-diabetic individuals who do not suffer from any kidney disease and inflammatory disease.

A variety of methods for determining the level of IL8 in a biological sample can be used. For example, the mRNA level of IL8 is measured by quantitative RT-PCR or by immunologically based analysis (such as ELISA and Western blot). For example, monoclonal antibody reagents for IL8 in enzyme-linked immunosorbent assay (ELISA) are described in U.S. Patent Nos. 6,133,426 and 6,468,532. The contents of each of these patents are incorporated herein by reference in their entirety.

In another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the urine IL-8 level of the subject is higher than the IL8 reference standard, wherein the reference standard is the average IL8 urine level of a statistically significant number of non-diabetic individuals who do not suffer from any renal disease and inflammatory disease. In one embodiment of any of the methods or uses, the elevated IL8 level means at least an increase of at least 10% compared to the reference IL8 level. In other embodiments of any of the methods, the elevated IL8 level means at least an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase or any increase between 10-100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to the reference IL-8 level.

In another embodiment of the above-mentioned purposes of the present invention, in combination with any of the foregoing embodiments, in the method or use, the subject has an IL8 expression level in a renal biopsy that is higher than an IL8 reference standard, wherein the reference standard is the average IL8 expression level in a renal biopsy from a non-diabetic individual who does not suffer from any renal disease and inflammatory disease. In another embodiment of the above-mentioned purposes of the present invention, in combination with any of the foregoing embodiments, in the method or use, the reference level of urine IL8 is the average urine IL8 level from a non-diabetic individual who does not suffer from any renal disease and inflammatory disease. For example, the average level is obtained from a group of 10-25 non-diabetic individuals who have never suffered from any renal disease and inflammatory disease. In another embodiment of the above-mentioned purposes of the present invention, in combination with any of the foregoing embodiments, in the method or use, the reference level of protein in urine is the average urine protein level from a non-diabetic individual who does not suffer from any renal disease and inflammatory disease. For example, the average level is obtained from a group of 10-25 non-diabetic individuals who have never suffered from any renal disease and inflammatory disease.

For example, in real life, it is expected that when an individual is diagnosed with diabetes but has not yet developed diabetic nephropathy (eg, in the case of normoproteinuria), the individual's IL8 level is further tested. When it has been demonstrated that the diabetic individual has an increased or elevated IL8 level, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A) to prevent the onset of diabetic nephropathy. In addition, when the diabetic individual develops early diabetic nephropathy, the individual's IL8 level is also tested. When it has been shown that this individual also has increased or elevated IL8 levels, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably Reparixin and/or Ladarixin, is then immediately administered prophylactically to treat, prevent and/or delay progression of diabetic nephropathy in the individual.

In another embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject's urine IL8 level is at least above 2.41 pg/ml.

In another embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject has a urine IL8 level above 2.41 pg/ml and has microalbuminuria.

As demonstrated in the experimental section below, this urinary IL8 level identifies patients at a stage of pathology in which IL8 plays a key role and thus responds to therapeutic treatment with IL8 inhibitors.

According to another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject's GFR (glomerular filtration rate) value is higher than 60 ml/min/1.73 m2, preferably higher than 90 ml/min/1.73 m2.

As demonstrated in the experimental part, IL8 has a pathogenic role in very early stages of pathology, when structural damage to the glomeruli is not yet apparent and the decline in GFR has not yet set in. Therefore, the above GRF values identify patients at a stage of pathology in which IL8 has a key role and therefore respond to therapeutic treatment with IL8 inhibitors.

As will be discussed in the experimental section, the inventors have also identified multiple single nucleotide polymorphisms of the CXCR1 receptor gene that are associated with the development of diabetic nephropathy.

Therefore, in another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

In yet another embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject has high HbA1C.

In yet another embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject is not overweight or obese.

In yet another embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject is overweight or obese.

In yet another embodiment of the above objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the subject is a human.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring the expression level of IL8 in a renal biopsy from a subject; (b) comparing the measured IL8 expression level with a reference standard; (c) when the measured IL8 level is higher than the IL8 reference standard, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably a compound selected from the group consisting of: R(-)-2- [(4-isobutylphenyl)propionic acid]-methanesulfonamide (hereinafter referred to as reparixin) or its salt, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or its salt, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A), wherein the reference standard is the average IL-8 expression level in renal biopsies from non-diabetic patients who do not suffer from any renal disease or inflammatory disease.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring urine IL8 levels in a subject; (b) comparing the measured IL8 levels with a reference standard; (c) when the measured IL8 levels are higher than the IL8 reference standard, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably a compound selected from the group consisting of: R(-)-2-[(4-iso [butylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or its salt, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or its salt, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A), wherein the reference standard is the average urine IL8 level of a statistically significant number of non-diabetic patients who do not suffer from any renal disease or inflammatory disease.

For example, in real life, it is expected that when a subject is diagnosed with diabetes but has not yet developed diabetic nephropathy, the individual's IL8 level is further tested and the measured IL8 level is compared to an IL8 reference standard. When it has been demonstrated that the diabetic subject has increased or elevated IL8 levels compared to the IL8 reference standard, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), is immediately administered prophylactically to prevent or delay the onset of diabetic nephropathy. In addition, when a diabetic subject develops early diabetic nephropathy, the subject's IL8 level is further tested, and the measured IL8 level is compared with an IL8 reference standard. When it has been demonstrated that the diabetic individual has an increased or elevated IL8 level relative to a reference IL8 level, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably the following compounds: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), to treat, prevent and/or delay the progression of diabetes in the diabetic subject.

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or development of diabetic nephropathy, the method comprising: (a) measuring the urine IL8 level in the subject; (b) when the measured urine IL8 level is above 2.41 pg/ml, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably a chemical selected from the following: Compounds: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring the level of protein in a urine sample obtained from the subject; (b) comparing the measured urine protein level with a urine protein reference standard; and (c) when the measured urine protein level is higher than the urine protein reference, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably a compound selected from the following: R(- )-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a lysine salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), wherein the reference is the average urine protein level of a statistically significant number of non-diabetic individuals who do not suffer from any kidney disease. In another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, the reference level of protein in urine is the average urine protein level from non-diabetic individuals who do not suffer from any kidney disease and inflammatory disease. For example, the average level is obtained from a population of 10-25 non-diabetic individuals who do not suffer from any kidney disease and inflammatory disease.

In practice, for example, when a subject is expected to be diagnosed with diabetes but has not yet developed diabetic nephropathy, the individual's urine protein level is further tested and the measured urine protein level is compared with a urine protein reference level. When it has been demonstrated that the diabetic subject has an increased or elevated urine protein level compared to a reference, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a lysine salt thereof, a sodium salt thereof (hereinafter referred to as DF2755A) to prevent or delay the onset of diabetic nephropathy. Furthermore, when a diabetic subject has developed early DN, the subject's urine protein level is further tested, and the measured urine protein level is compared with a urine protein reference level. When it has been demonstrated that the diabetic individual has an increased or elevated urine protein level compared to a urine protein reference level, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A) is immediately administered to prevent and/or delay the progression of diabetic nephropathy in diabetic subjects.

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring the albumin excretion rate in urine from the subject; (b) when the measured albumin excretion rate is 30 to 300 mg/day, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, even more preferably selected from the following Compounds: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or its salt, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or its salt, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A).

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) determining whether a subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514; (b) when the subject has at least one of the SNPs, administering to the subject an IL8 inhibitor, preferably a CXCR1 and/or C XCR2 inhibitors, more preferably CXCR1 and CXCR2 inhibitors, even more preferably compounds selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In real life practice, for example, it is expected that when a subject has been diagnosed with diabetes but has not yet developed diabetic nephropathy, the genome of the individual is further tested for the SNPs. When it has been confirmed that the diabetic subject has at least one of the SNPs, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a salt thereof, a sodium salt thereof (hereinafter referred to as DF2755A) to prevent or delay the onset of DN. In addition, when the diabetic subject has developed early diabetic nephropathy, the individual genome is further tested for the SNPs. When it has been demonstrated that the diabetic subject has at least one of the SNPs, an IL8 inhibitor is immediately administered, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as Reparixin); R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as Ladarixin); and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its sodium salt (hereinafter referred to as DF2755A), to prevent and/or treat and/or delay the onset or progression of diabetic nephropathy in the subject.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or developing diabetic nephropathy, the method comprising: (a) determining whether the subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514; (b) measuring urine IL8 levels in the subject; (c) comparing the measured IL8 levels to a reference; (d) when the subject has at least one of the SNPs and has increased IL8 relative to the reference, administering to the subject an IL8 inhibitor, preferably a CXCR1 inhibitor; R1 and/or CXCR2 inhibitors, more preferably CXCR1 and CXCR2 inhibitors, even more preferably compounds selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), wherein the reference is the average urine IL8 level of a statistically significant number of non-diabetic individuals who do not suffer from any renal disease or inflammatory disease.

In another embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) determining whether the subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514; (b) measuring the level of IL8 expression in a renal biopsy from the subject; (c) comparing the measured IL8 expression level to a reference; and (d) administering to the subject when the subject has at least one of the SNPs and has increased IL8 expression relative to the reference. IL8 inhibitors, preferably CXCR1 and/or CXCR2 inhibitors, more preferably CXCR1 and CXCR2 inhibitors, even more preferably compounds selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as 1adarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), wherein the reference is the average renal IL8 expression level in non-diabetic individuals who do not suffer from renal disease or inflammatory diseases.

In practice, for example, it is expected that when a subject has been diagnosed with diabetes or has a diabetic condition but has not yet developed DN, the individual's genome is further tested for the SNPs, and the individual's IL8 level is further tested, and the measured IL8 level is compared with the reference IL8. When it has been demonstrated that the diabetic subject has at least one of the SNPs and has an increased or elevated IL8 level compared to the reference IL8, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), to prevent or delay the onset of diabetic nephropathy. Furthermore, when the diabetic subject has developed early diabetic nephropathy, the genome of the individual is further tested for the SNP, and the IL8 level of the individual is further tested, and the measured IL8 level is compared with a reference IL8. When it has been demonstrated that the diabetic subject has at least one of the SNPs and has an increased or elevated IL8 level relative to the reference IL-8 level, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), to treat, prevent and/or delay the progression of diabetic nephropathy in the diabetic subject.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) determining whether the subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514; (b) measuring the urine protein level in the subject; (c) comparing the measured protein level to a protein reference; (d) when the subject has at least one of the SNPs and the protein in the urine is When the amount of IL-8 increases, the subject is administered an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). In one embodiment, the method further comprises obtaining a urine sample from the subject for protein level analysis. In practice, it is expected that when a subject is diagnosed with diabetes but has not yet developed DN, the individual's genome is further tested for the SNPs, and the individual's urine protein level is further tested, and the measured urine protein level is compared with a urine protein reference level. When it has been demonstrated that the diabetic subject has at least one of the SNPs and has an increased or elevated urine protein level relative to the reference level, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-l,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), to prevent or delay the onset of DN. Furthermore, when a diabetic subject develops early diabetic nephropathy, the genome of the individual is further tested for the SNP, and the urine protein level of the individual is further tested, and the measured urine protein level is compared with a urine protein reference level. When it has been demonstrated that the diabetic subject has at least one of the SNPs and has an increased or elevated urine protein level relative to a urine protein reference level, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), is immediately administered prophylactically to treat, prevent and/or delay the progression of diabetic nephropathy in the diabetic subject.

In one embodiment of the first object of the present invention, in combination with any of the foregoing embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) measuring the level of IL8 in a sample obtained from the subject; (b) comparing the measured IL8 level with a reference; (c) measuring the protein level in a sample obtained from the subject; (d) comparing the measured protein level with a protein reference, and (e) administering Reparixin and/or Ladarixin to the subject when the IL8 and protein in the urine of the subject increase. In one embodiment, the sample for protein level analysis is a urine sample. In one embodiment, the method further comprises obtaining a urine sample from the subject for protein level analysis. In one embodiment, the sample for IL8 level analysis is a urine, serum, blood or plasma sample. In one embodiment, the method further comprises obtaining a sample from the subject for IL8 analysis. In practice, when a subject is diagnosed with diabetes but has not yet developed DN, the individual is further tested for IL8 levels and compared with a reference IL8, and further tested for urine protein levels and compared with a urine protein reference level. When it has been demonstrated that the diabetic subject has an increased or elevated IL8 level compared to a reference IL8, and also demonstrated an increased or elevated urine protein level relative to a urine protein reference level, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), to prevent or delay the onset of DN. Furthermore, when the diabetic subject has developed early DN, the subject is further tested for IL8 level and compared with an IL8 reference, and is further tested for urine protein level and compared with a urine protein reference level. When it has been demonstrated that the diabetic subject has an increased or elevated IL8 level compared to a reference IL8, and also demonstrated an increased or elevated urine protein level relative to a urine protein reference level, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A), is immediately administered prophylactically to treat, prevent and/or delay the progression of DN in the diabetic subject.

In one embodiment of the first object of the present invention, in combination with any of the preceding embodiments, provided herein is a method for treating diabetic nephropathy in a subject or preventing, reducing the risk of, or delaying the onset or progression of diabetic nephropathy, the method comprising: (a) determining whether the subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514; (b) measuring the level of IL8 in a sample obtained from the subject; (c) comparing the measured level of IL8 with a reference; (d) measuring the level of protein in a sample obtained from the subject; (e) comparing the measured protein level with a protein reference, and (f) when the subject has the At least one of the above SNPs, when there is increased protein in the urine and increased IL8, an IL8 inhibitor is administered to the subject, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). In one embodiment, the sample for protein level analysis is a urine sample. In one embodiment, the method further comprises obtaining a urine sample from the subject for protein level analysis. In one embodiment, the sample for IL8 level analysis is urine, serum, blood or plasma sample. In one embodiment, the method also includes obtaining a sample from the subject for IL8 analysis. In real life practice, when the subject is diagnosed with diabetes but has not yet developed into diabetic nephropathy, the SNP of the individual genome is further tested, and the IL8 level of the individual is further tested and the urine protein level is tested. The measured IL8 level is compared with reference to IL8, and the measured urine protein level is compared with the urine protein reference level. When it has been proved that the diabetic subject has at least one of the SNPs, and it is proved that the IL8 level with an increase or increase compared with the reference to IL8, and the urine protein level with an increase or increase relative to the urine protein reference level, then immediately prophylactically administer Reparixin and/or Ladarixin to prevent the onset of diabetic nephropathy. In addition, when the diabetic subject has developed into early diabetic nephropathy, the SNP of the individual genome is further tested, and the IL8 level of the individual is further tested and the urine protein level is tested. The measured IL8 level is compared with reference to IL8, and the measured urine protein level is compared with the urine protein reference level. When the diabetic subject has been confirmed to have at least one of the SNPs, and has been confirmed to have an increased or elevated IL8 level compared to a reference IL8, and an increased or elevated urine protein level relative to a urine protein reference level, an IL8 inhibitor is immediately administered prophylactically, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as Ripa R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methylsulfonylpropionamide (hereinafter referred to as ladarixin) or its salt, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A), to prevent and/or delay the progression of diabetic nephropathy in the diabetic subject. It is expected that early application of IL8 inhibitors will prolong the duration of end-stage renal disease or chronic renal failure in these individuals. The IL-8 inhibitors are preferably CXCR1 and/or CXCR2 inhibitors, more preferably CXCR1 and CXCR2 inhibitors, and even more preferably compounds selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-l,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably its sodium salt (hereinafter referred to as DF2755A).

In one embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an effective amount of an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propanoic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an effective amount of an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In another embodiment of the above-mentioned objects of the present invention, in combination with any of the above-mentioned embodiments, in the method or use, a composition comprising an effective amount of an IL8 inhibitor is administered to a subject, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4)-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). Preferably, the composition comprises a pharmaceutically acceptable carrier. Preferably, the composition further comprises at least one other active molecule for diabetes and/or metabolic syndrome and/or cardiovascular disease and/or hypertension.

For example, diabetes is usually treated with a drug or a combination of drugs, including sulfonylureas, meglitinides, biguanides, thiazolidinediones, α-glucosidase inhibitors and DPP-4 inhibitors. In one embodiment of any of the methods, an IL8 inhibitor is administered together with at least one active molecule to treat diabetes, and the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as Reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy]- [4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl]propanoic acid (hereinafter referred to as DF2755Y) or its salt, preferably its sodium salt (hereinafter referred to as DF2755A), wherein the active molecule is preferably selected from sulfonylureas, meglitinides, biguanides, thiazolidinediones, α-glucosidase inhibitors and DPP-4 inhibitors.

For example, the at least one other active molecule is insulin, angiotensin converting enzyme (ACE) inhibitor, angiotensin-II receptor antagonist (AIIRA), a drug or agent that lowers blood HbA1c (e.g., telenzepin and sertraline described in U.S. Pat. No. 8,440,655, the contents of which are incorporated herein by reference in their entirety). In one embodiment of any of the methods, an IL8 inhibitor is administered together with at least one active ingredient including an ACE inhibitor, AIIRA, telenzepin and sertraline, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as Reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as Ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor is administered in combination with one or more antidiabetic agents, and the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In another embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor is administered together with at least one other active molecule for diabetes and/or metabolic syndrome and/or cardiovascular disease and/or hypertension, and the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(2-)trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In one embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor is systemically administered, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In one embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In one embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In one embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In one embodiment of the above-mentioned objects of the present invention, in combination with any of the preceding embodiments, in the method or use, an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In one embodiment, the IL8 inhibitor or its pharmaceutical composition is formulated for systemic delivery, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). In an optional embodiment, the IL8 inhibitor and its pharmaceutical composition can be formulated for delivery to a specific organ, such as but not limited to the kidney. The IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). In an optional embodiment, the IL8 inhibitor or its pharmaceutical composition can be formulated for application by inhalation of an aerosol into the lungs, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). Optionally, the IL8 inhibitor or its pharmaceutical composition can be formulated for transdermal delivery, such as a skin patch, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). In some embodiments, an IL8 inhibitor or a pharmaceutical composition thereof can be enteric coated and formulated for oral delivery, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). In some embodiments, the IL8 inhibitor or its pharmaceutical composition can be encapsulated into liposomes or nanoparticles and formulated for sustained release delivery in vivo. The IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). Also contemplated are sustained release formulations comprising an IL8 inhibitor or a pharmaceutical composition thereof, the IL8 inhibitor being preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the group consisting of: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A). For example, a sustained release formulation for once-weekly administration. Optionally, the IL8 inhibitor or its pharmaceutical composition can be formulated for targeted delivery, for example, encapsulated into designed liposomes or nanoparticles, which are characterized by a targeting portion on the liposomes or nanoparticles, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as relixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

The IL8 inhibitor or its pharmaceutical composition can be formulated and administered by any known route. For example, the IL8 inhibitor and its composition can be administered by mucosal, pulmonary, topical or other local or systemic routes (e.g., enteral and parenteral). The IL8 inhibitor can be administered by any convenient route, for example by infusion or bolus injection, absorbed through epithelial or mucocutaneous linings (e.g. oral mucosa, rectal and intestinal mucosa, etc.), and can be used together with other biologically active agents, the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as reparixin) or a salt thereof, preferably its lysine salt; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably its sodium salt; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably its sodium salt (hereinafter referred to as DF2755A).

Administration routes include, but are not limited to, aerosol, direct injection, intradermal, transdermal (e.g., in the form of a sustained-release polymer), intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, topical, oral, transmucosal, buccal, rectal, vaginal, transdermal, intranasal, and parenteral routes. "Parenteral" refers to routes of administration typically associated with injection, including but not limited to intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intrahepatic, intraorgan, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Any other therapeutically effective route of administration can be used, such as infusion or bolus injection, absorbed by the epithelium or mucocutaneous lining. In a number of embodiments, administration can be inhaled into the lungs via aerosol administration, such as by atomization. Administration can also be systemic or local.

For example, an IL8 inhibitor or its pharmaceutical composition can be administered as a formulation suitable for systemic delivery. In some embodiments, an IL8 inhibitor or its pharmaceutical composition can be administered as a formulation suitable for delivery to a specific organ such as, but not limited to, the kidney, wherein the IL8 inhibitor is preferably a CXCR1 and/or CXCR2 inhibitor, more preferably a CXCR1 and CXCR2 inhibitor, and even more preferably a compound selected from the following: R(-)-2-[(4-isobutylphenyl)propionyl]-methanesulfonamide (hereinafter referred to as Reparixin) or a salt thereof, preferably a lysine salt thereof; R(-)-2-[(4'-trifluoromethanesulfonyloxy)phenyl]-N-methanesulfonylpropionamide (hereinafter referred to as ladarixin) or a salt thereof, preferably a sodium salt thereof; and (2S)-2-(4-{[4-(trifluoromethyl)-1,3-thiazol-2-yl]amino}phenyl)propionic acid (hereinafter referred to as DF2755Y) or a salt thereof, preferably a sodium salt thereof (hereinafter referred to as DF2755A).

In addition, the IL8 inhibitor or pharmaceutical composition thereof can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants (eg, glycerides), excipients (eg, lactose), carriers, diluents, and vehicles.

An IL8 inhibitor or a pharmaceutical composition thereof can be therapeutically administered to a subject prior to, concurrently with, or after administration of at least one therapy for diabetes, metabolic syndrome, cardiovascular disease, and hypertension.

For parenteral (e.g., intravenous, subcutaneous, intramuscular) administration, the IL8 inhibitor or its pharmaceutical composition can be formulated as a solution, suspension, emulsion or lyophilized powder combined with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by conventional techniques.

The dosage administered to a subject will vary according to a variety of factors, including the pharmacodynamic characteristics of the particular inhibitor and its mode and route of administration; the size, age, sex, health, weight and diet of the recipient; the nature and extent of the symptoms of the disease being treated, kind of concurrent treatment, frequency of treatment and the desired effect.

Typically, the daily dose of the IL8 inhibitor may be about 1 to 100 mg/kg body weight, preferably 5 to 80 mg/kg/day. Preferably, the desired results may be obtained by oral administration in divided doses of 1 to 5 times a day or by continuous infusion for 5 to 10 days in one or more cycles. The second or subsequent administration may be a dose that is the same, less than, or greater than the initial or previous dose administered to the individual.

The second or subsequent administration is preferably administered immediately before or during a relapse or sudden onset of disease or disease symptoms. For example, the second and subsequent administrations can be given about one day to 30 weeks after the previous administration. As needed, two, three, four or more administrations can be delivered to an individual in total.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Efficacy testing can be performed during treatment using the methods described herein.Before treatment begins, and then at specific time periods after treatment begins, measures of the severity of a number of symptoms associated with the specific disease are noted.

The precise dose to be used in the formulation of the pharmaceutical agent will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The methods described herein can be used to perform efficacy testing during treatment. Before treatment begins, then at a specific time period after treatment begins, note the measurement of the severity of many symptoms associated with a specific disease. For example, during treatment.

The skilled person will appreciate that certain factors may affect the dosage and time required for effective treatment of the subject, including but not limited to the severity of the disease or condition, prior treatment, the general health and/or age of the subject, and other diseases present. The dosage level may also depend on the degree of nephropathy, the severity of the symptoms, and the sensitivity of the subject to side effects. Treating the subject with a therapeutically effective dose may include a single treatment or a series of treatments. As known in the art, or as described herein, the effective dose of IL8 inhibitors and the estimation of the in vivo half-life can be carried out using conventional methods or based on in vivo testing using a suitable animal model. Those skilled in the art can easily determine the preferred dosage in a variety of ways.

Some embodiments of the technology described herein may be defined according to any of the following numbered paragraphs:

[1] A method for preventing the onset of diabetic nephropathy or the progression of diabetic nephropathy (DN) in a subject in need thereof, comprising administering to the subject diagnosed with diabetes an IL8 inhibitor, preferably a CXCR1 and/or CXCR2 inhibitor, more preferably Reparixin and/or Ladarixin.

[2] A method for preventing the onset of diabetic nephropathy (DN) or the progression of diabetic nephropathy in a subject in need thereof, comprising administering Reparixin and/or Ladarixin to the subject who has been diagnosed with diabetes and has elevated levels of IL8.

[3] The method of paragraph 1 or 2, wherein the diabetes is type 1 diabetes (T1D).

[4] The method of paragraph 1 or 2, wherein the diabetes is type 2 diabetes (T2D).

[5] The method of any of paragraphs 1-4, wherein the subject has normal proteinuria.

[6] The method of any of paragraphs 1-4, wherein the subject has increased proteinuria.

[7] The method of any of paragraphs 1-6, wherein the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[8] The method of any of paragraphs 1-7, further comprising measuring protein levels in a urine sample from the subject.

[9] The method of any one of paragraphs 1-8, further comprising selecting a subject having proteinuria.

[10] The method of any one of paragraphs 1-9, further comprising obtaining a urine sample from the subject for analysis of urine protein levels.

[11] The method of any of paragraphs 8-10, further comprising comparing the measured urine protein level with a urine protein reference.

[12] The method as described in paragraph 11, wherein the urine protein reference is the protein level in a urine sample obtained from a normal healthy subject who does not suffer from any kidney disease.

[13] The method of any of paragraphs 1-12, further comprising measuring the level of IL8 in a sample obtained from the subject.

[14] The method of paragraph 13, wherein the sample is a urine sample.

[15] The method of paragraph 13, wherein the sample is a serum, blood or plasma sample.

[16] The method of any of paragraphs 13-15, further comprising comparing the measured IL8 level to an IL8 reference.

[17] The method of paragraph 16, wherein the IL8 reference is the IL8 level in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

[18] The method of any of paragraphs 1-17, further comprising determining whether the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[19] A method for preventing the onset or progression of diabetic nephropathy (DN) in a subject diagnosed with diabetes and microalbuminuria, the method comprising:

a) measuring the level of IL8 in a sample obtained from the subject; and

b) administering Reparixin and/or Ladarixin to the subject when the measured IL8 level is at least above 2.41 pg/ml.

[20] The method of paragraph 19, wherein the diabetes is type 1 diabetes (T1D).

[21] The method of paragraph 19, wherein the diabetes is type 2 diabetes (T2D).

[22] The method of any of paragraphs 19-21, wherein the sample is a urine sample.

[23] The method of any of paragraphs 19-21, wherein the sample is a serum, blood or plasma sample.

[24] The method of any of paragraphs 19-23, wherein the subject has normal proteinuria.

[25] The method of any of paragraphs 19-23, wherein the subject has increased proteinuria.

[26] The method of any of paragraphs 19-25, wherein the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[27] The method of any of paragraphs 19-26, further comprising measuring protein levels in a urine sample from the subject.

[28] The method of any of paragraphs 19-27, further comprising obtaining a urine sample from the subject for analysis of protein levels.

[29] The method of any of paragraphs 19-28, further comprising comparing the urine protein level to a urine protein reference.

[30] The method as described in any of paragraphs 19-29, wherein the urine protein reference is the protein level in a urine sample obtained from a normal healthy subject not suffering from any kidney disease.

[31] The method as described in any of paragraphs 19-30, wherein the IL8 reference is the IL8 level in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

[32] The method of any of paragraphs 19-31, further comprising determining whether the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[33] A method for preventing the onset of diabetic nephropathy (DN) or the progression of diabetic nephropathy (DN) in a subject diagnosed with diabetes, the method comprising:

a) measuring the level of protein in a urine sample obtained from the subject;

b) comparing the measured urine protein level to a urine protein reference; and

c) administering Reparixin and/or Ladarixin to the subject when the measured urine protein level is higher than the urine protein reference.

[34] The method of paragraph 33, wherein the diabetes is type 1 diabetes (T1D).

[35] The method of paragraph 33, wherein the diabetes is type 2 diabetes (T2D).

[36] The method of any of paragraphs 33-35, wherein the urine protein reference is the protein level in a urine sample obtained from a normal healthy subject not suffering from any kidney disease.

[37] The method of any of paragraphs 33-35, further comprising measuring the level of IL8 in a sample obtained from the subject.

[38] The method of paragraph 37, wherein the sample is a urine sample.

[39] The method of paragraph 37, wherein the sample is a serum, blood or plasma sample.

[40] The method of any of paragraphs 33-39, further comprising comparing the measured IL8 level to an IL8 reference.

[41] The method of paragraph 40, wherein the IL8 reference is the IL8 level in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

[42] The method of any of paragraphs 33-41, further comprising determining whether the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[43] A method for preventing the onset of diabetic nephropathy (DN) or the progression of diabetic nephropathy (DN) in a subject diagnosed with diabetes, the method comprising:

a) determining whether the subject has at least one of the following single nucleotide polymorphisms (SNPs) at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514;

b) administering Reparixin and/or Ladarixin to said subject when said subject has at least one of said SNPs.

[44] The method of paragraph 43, wherein the diabetes is type 1 diabetes (T1D).

[45] The method of paragraph 43, wherein the diabetes is type 2 diabetes (T2D).

[46] The method of any of paragraphs 43-45, wherein the subject has normal proteinuria.

[47] The method of any of paragraphs 43-45, wherein the subject has increased proteinuria.

[48] The method of any of paragraphs 43-47, further comprising measuring protein levels in a urine sample from the subject.

[49] The method of any of paragraphs 43-48, further comprising obtaining a urine sample from the subject for analysis of urine protein levels.

[50] The method of paragraph 49, further comprising comparing the measured urine protein level to a urine protein reference.

[51] The method of paragraph 50, wherein the urine protein reference is the protein level in a urine sample obtained from a normal healthy subject not suffering from any kidney disease.

[52] The method of any of paragraphs 43-51, further comprising measuring the level of IL8 in a sample obtained from the subject.

[53] The method of paragraph 52, wherein the sample is a urine sample.

[54] The method of paragraph 52, wherein the sample is a serum, blood or plasma sample.

[55] The method of any of paragraphs 52-54, further comprising comparing the measured IL8 level to an IL8 reference.

[56] The method of paragraph 55, wherein the IL8 reference is the IL8 level in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

[57] A method of treating diabetic nephropathy (DN) in a subject in need of such treatment, the method comprising administering to the subject Reparixin and/or Ladarixin.

[58] The method of paragraph 57, wherein the subject has type 1 diabetes (T1D).

[59] The method of paragraph 57, wherein the subject has type 2 diabetes (T2D).

[60] The method of any of paragraphs 57-59, wherein the subject has elevated IL8 levels.

[61] The method of any of paragraphs 57-60, wherein the subject has normal proteinuria.

[62] The method of any of paragraphs 57-61, wherein the subject has increased proteinuria.

[63] The method of paragraphs 57-62, further comprising measuring the level of IL8 in a sample obtained from the subject.

[64] The method of paragraph 63, wherein the sample is a urine sample.

[65] The method of paragraph 63, wherein the sample is a serum, blood or plasma sample.

[66] The method of any of paragraphs 63-65, further comprising comparing the measured IL8 level to an IL8 reference.

[67] The method of paragraph 66, wherein the IL8 reference is the IL8 level in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

[68] The method of any of paragraphs 57-67, wherein the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[69] The method of paragraphs 57-68, further comprising determining whether the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

[70] The method of any of paragraphs 1-69, wherein the subject has a glomerular filtration rate (GFR) value greater than 60 ml/min/1.73 m ² .

[71] The method of paragraph 70, wherein the subject has a glomerular filtration rate value greater than 90 ml/min/1.73 m ² .

[72] The method of any of paragraphs 1-71, wherein the subject's urine IL8 level is greater than 2.4 lpg/ml.

[73] The method of any of paragraphs 1-71, wherein the subject has a measured albumin excretion rate of 30 to 300 mg per day.

[74] A method of treatment, comprising:

a) determining the level of IL8 in a urine sample from the subject; and

b) administering to the subject an effective amount of Reparixin and/or Ladarixin when the IL8 level is at least 3-fold higher than the reference level.

[75] The method of paragraph 74 further comprises diagnosing the subject as having diabetes.

[76] The method of paragraph 74, wherein the sample is a urine sample.

[77] The method of paragraph 63, wherein the sample is a serum, blood or plasma sample.

[78] The method of paragraph 74, wherein the reference level is the IL8 level in a corresponding sample obtained from a normal healthy subject not suffering from any kidney disease.

[79] A method for treating hyperglycemia, the method comprising:

a) diagnosing patients with hyperglycemia; and

b) administering to said patient an effective amount of Reparixin and/or Ladarixin.

[80] A method for treating hyperglycemia, the method comprising: administering an effective amount of Reparixin and/or Ladarixin to a patient in need thereof.

[81] A composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for use in treating diabetic nephropathy or preventing or reducing the risk or delaying the onset or progression of diabetic nephropathy in a subject.

[82] A composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for use in the preparation of a medicament for treating diabetic nephropathy or preventing or reducing the risk or delaying the onset or progression of diabetic nephropathy in a subject.

[83] Use of a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin for treating diabetic nephropathy or preventing or reducing the risk or delaying the onset or progression of diabetic nephropathy in a subject.

[84] Use of a composition comprising, consisting of, or consisting essentially of Reparixin and/or Ladarixin in the preparation of a medicament for treating diabetic nephropathy or preventing or reducing the risk or delaying the onset or progression of diabetic nephropathy in a subject.

Embodiments of the present invention are further illustrated by the following examples, which should not be construed as limiting.The contents of all references cited throughout this application, as well as the figures and tables, are incorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The references cited herein and the entire specification are incorporated herein by reference.

Example

Introduction

Among the various complications of diabetes, chronic kidney disease (CKD) has been identified as the complication with the highest burden on daily life and economic costs. CKD increases the risk of premature death and end-stage renal disease (ESRD)1. The incidence of end-stage renal disease (ESRD) caused by diabetes has been increasing over the past two decades. In more than one-third of patients in the Western world, diabetes is the main cause of ESRD2. More than 5% of newly diagnosed type 2 diabetes (T2D) patients already have diabetic nephropathy, and an additional 30% to 40% will develop diabetic nephropathy (DN), usually within 10 years of ^{diagnosis3,4,5} .

Patients with T2D have altered immune systems. Elevated levels of cytokines, chemokines, and acute phase proteins have been described in these patients6,7; altered immunological profiles have been shown to increase apoptosis and tissue fibrosis8. Patients with type 1 diabetes (T1D) have 6- to 7-fold higher urinary IL8 levels compared with controls9. Furthermore, among T1D patients with albuminuria, those with the highest baseline urinary IL8 levels had more rapid renal decline.

IL8 is a chemokine that can be produced by leukocytes such as monocytes 10, T lymphocytes, macrophages 11, or by non-leukocyte populations such as endothelial cells 12, podocytes 13, proximal tubular epithelial cells 14. This chemokine has two receptors, CXCR-1 and CXCR-2, which are expressed by leukocytes such as neutrophils, monocytes, CD8 T cells, mast cells, natural killer cells, and also by non-leukocytes such as endothelial cells 12, podocytes 13, fibroblasts 11.

In patients with diabetes, hyperglycemia can trigger the production of IL8, thereby stimulating CXCR1/2 to be expressed in an auto- and paracrine manner. The activation of CXCR1/2 diffuses in podocytes and endothelial cells through the CXCR1/2 cytoplasmic tail, which determines the inactivation of α3β1-integrin by competing with the cytoplasmic tail of α3β1-integrin for binding to talin (which is essential for the activity of α3β1-integrin; Figure 6). This competitive binding leads to the inactivation of paxillin, accompanied by the loss of podocyte physiological structure and adhesion to the glomerular basement membrane. CXCR1/2 activation also leads to the activation of the mTor pathway, causing metabolic changes and oxidative damage. In short, these pathological changes ultimately lead to abnormal podocyte structure and function and the development of proteinuria (Figures 7A-7E and Figures 8A-8F). The invention described herein shows that neutralizing IL8 signal transduction with clinically available CXCR1/2 antagonists will reduce the extent of podocyte disease in vitro and the progression of renal injury in vivo, and provide new therapeutic tools for DN.

Example 1

In vivo, CXCR2 and KC expression increased gradually in the glomeruli of STZ-induced C57BL/6 diabetic mice and were localized in endothelial cells and podocytes.

In a model of DN db/db diabetic mice, the in vivo expression of KC (mouse homolog of human IL8) and its receptor CXCR2 (mouse homolog of human CXCR2) in STZ-induced C57BL/6 diabetic mice was evaluated. Diabetes is defined in this article as blood glucose levels >250 mg/d1 for 3 consecutive days. Glucose levels in serum were measured using a BD Logic blood glucose meter (Becton Dickinson, FranklinLakes, NJ). At week 7, db/db mice presented hyperglycemia but not DN. C57BL/6 mice and C57BL/6J Lepdb/db were obtained from Jackson Laboratories (Bar Harbor, Maine). Mice were raised in a pathogen-free environment; water and food were provided ad libitum. All mice were male and were cared for and used in accordance with the Animal Care and Husbandry Guidelines of Boston Children's Hospital and Harvard Medical School. The Institutional Animal Care and Use Committee approved the protocol.

Kidneys from 8-week-old, 12-week-old, and 25-week-old db/db mice were surgically removed using standard techniques. For standard light microscopy, kidneys were fixed in 4% buffered paraformaldehyde (PFA), dehydrated, and embedded in paraffin. Images from Periodic Acid Schiff (PAS) and Trichrome staining were recorded using AxioVision software 4.3. Evaluation of glomerular mesangial matrix was performed using a macro built based on the AxioVision analysis module (Carl Zeiss Spa, Thomwood, NY). Glomeruli were identified as regions of interest (ROIs) and mesangial highlights were made using a color thresholding scheme. Binary images were then generated, and the percentage of mesangial area in the glomerular area was automatically calculated. Image acquisition was performed using a Zeiss Axioscope 40FL microscope and an AxioCam MRc5 digital camera (Carl Zeiss SpA). Images were recorded using AxioVision software 4.3, and the results were analyzed using the AxioVision analysis module (Carl Zeiss SpA).

KC and CXCR2 expression increased at the glomerular level in diabetic mice at 12 weeks of age, and expression peaked at 28 weeks of age (data not shown). In addition, at 28 weeks, CXCR2 and KC expression colocalized with CD31 (endothelial cell marker) and synaptopodin (podocyte-specific marker) at the glomerular level in db/db C57BL/6 diabetic mice (data not shown). In contrast, kidneys obtained from 7-week (baseline) non-diabetic C57BL/6 control mice did not express CXCR2 and KC.

Example 2

Blockade of the KC/CXCR2 axis with reparixin prevented increased urinary albumin excretion (UAE) and attenuated mesangial expansion in db/db mice.

To evaluate the potential role of IL8 blockade in DN progression, 7-week-old db/db mice were treated with Reparixin, 15 mg/kg (ip), twice a day, for 18 weeks (until 25 weeks of age). Briefly, animals were housed in metabolic cages (Nalgene) to separate excreta and urine in a light-controlled environment, and water was provided ad libitum. Sample collection tubes were passed under the cages and through a small hole in the bottom of the light-controlled environment.

Urine samples from db/db mice were collected by metabolic cages at weeks 8, 12, and 25. The level of cytokine IL8 was determined using BeadLyte Mouse Multi-cytokine Beadmaster Kit (Millipore, Billerica, MA) according to the manufacturer's protocol. In brief, the supernatant was incubated with beads conjugated to IL8 alone for a specified time, followed by incubation with biotinylated reporter molecules and streptavidin-phycoerythrin solution for 30 minutes. Sample cytokine levels were measured using a Luminex100 reader (Luminex Corporation, Austin, TX).

While urinary albumin excretion was increased in untreated control mice, in mice treated with Reparixin, urinary albumin levels remained stable over time (Figures 1 and 11B) and were significantly lower than control diabetic db/db mice at 25 weeks of age, showing a significant time-treatment interaction at the most recent time point (Reparixin treated-25 weeks = 211.1 ± 24.4 vs. control-25 weeks = 353.8 ± 99.4 ug/ml, p < 0.01; Figure 1). No effects on glycemic control were observed in the treatment groups (Figure 11A). Renal biopsies from the above animals were subjected to histopathological examination, and Reparixin treatment showed a protective effect on the kidneys of 25-week-old db/db mice, with a significant reduction in mesangial expansion in treated animals compared to control mice (data not shown).

Example 3

IL8 stimulation caused a loss of stress fibers, a dose-dependent increase in cortical actin, and cellular blebbing in podocytes in vitro.

Human podocytes carrying a transgenic variant of the SV40 T antigen thermosensitive (ts58A) were cultured, allowed to respond to interferon-γ for proliferation, and grown to 80% confluence at 33°C (designated as day 0). The cells were then heat-shifted to 37°C, causing inactivation of the SV40 T antigen and cessation of cell replication. Podocytes were cultured for 5 days at different glucose concentrations (normal glucose: 5mM [NG]; high glucose: 30mM [HG]). Mannitol was used as an osmotic control for high glucose (mannitol 20mM + glucose 10mM) (Figures 10A and 10B).

Podocytes were grown on round glass coverslips (VWR, Radnor, PA), fixed with paraformaldehyde, and then permeabilized with 0.3% Triton X-100 (Fisher Scientific, Waltham, MA). Cells were incubated with rhodamine phalloidin (Invitrogen, Carlsbad, CA) to label the F-actin network and visualize stress fibers. Stress fiber formation was assessed using standard fluorescence microscopy. The percentage of cells containing intact actin filaments was assessed by manual cell counting.

In vitro treatment with 100 nM IL8 induced loss of stress fibers in human podocytes (measured by phalloidin staining; control vs. IL8, p < 0.05; Figure 2A), a dose-dependent increase in cortical actin, cellular blebbing, and synaptopodin c expression (control vs. IL8, p < 0.05; Figure 2B), both under NG and HG (data not shown) conditions; the latter acting synergistically with IL8. These data support the hypothesis that IL8 induces direct podocyte injury.

Interestingly, treatment with reparixin at a dose of 100 μM was able to rescue podocytes from IL8-induced damage (loss of stress fibers; increase in cortical actin; cell blebbing and loss of synaptic protein expression) in both NG (data not shown) and HG (data not shown; and Figures 2A, 2B, 10A, and 10B).

Example 4

IL8 is expressed at the glomerular level in a subset of patients with T2D and DN and colocalizes with CD-31 and synaptopodin.

As described above, the expression of IL8 in renal biopsies of 30 patients with type 2 diabetes (T2D) and DKD at different stages of severity (from less severe to increasingly severe: mesangial expansion; nodular transformation; and glomerulosclerosis) and from control individuals (surgical removal of the kidney affected by cancer) (data not shown) was measured by immunohistochemical analysis. Histological data from this group were published by Fiorina et al. in 2013. Informed consent was approved by the Institutional Review Board of Hospital San Carlo (Milan, Italy) and/or the Institutional Review Board of Azienda Ospedaliera di Parma, Parma, Italy before the patient signed it. Medical history records were obtained for each patient, and renal function data were obtained from serum and urine samples. As a control, unaltered renal pedicle histological samples from patients with renal cancer who underwent unilateral nephrectomy (n = 10) were used.

Materials for conventional light microscopy staining were fixed in 4% buffered paraformaldehyde (PFA), dehydrated and paraffin embedded. Images from PAS and Trichrome staining were recorded using AxioVision software 4.3, and the evaluation of mesangial matrix was performed electronically based on a macro established based on AxioVision analysis module (Carl Zeiss SpA, Thornwood, NY). In brief, glomeruli were identified as regions of interest (ROIs), and mesangium was highlighted by a color thresholding program. Binary images were then generated, and mesangium was automatically calculated as a percentage of glomerular area. Image acquisition was performed using a Zeiss Axioscope 40FL microscope and an AxioCam MRc5 digital camera (Carl Zeiss SpA). Images were recorded using AxioVision software 4.3, and the results were analyzed using the AxioVision analysis module (Carl Zeiss SpA). Anti-human IL8 antibodies were obtained from Abcam (Cambridge, MA). The staining of 40 glomeruli per sample was evaluated as the number of positive points per glomerulus. As shown in Figures 3A and 3B, a correlation between the stage of renal injury and IL8 expression was found. It was found that IL8 expression peaked in the early injury stage and gradually decreased after the onset of renal parenchymal cellularity loss and fibrosis (data not shown; Figures 7A and 7B). During the mesangial expression period, the expression of IL8 in the glomerulus was the highest (data not shown; Figure 3A and Table 2): mean GFR = 91.43 ± 7.74m1/min/ ^1.73m2 ; IL8 histopathological score: 3 ± 0.0 arbitrary unit (AU)). A continuous decrease in IL8 staining was observed with progression of glomerular lesions toward nodular transformation (data not shown; FIG. 3A and Table 2: mean GFR = 62.29 ± 6.75 ml/min/1.73 m ² ; IL8 histopathology score: 1.5 ± 0.41 arbitrary units (AU)) and glomerular sclerosis (data not shown; FIG. 3A ; and Table 2: mean GFR = 48.25 ± 8.52 ml/min/1.73 m ² ; IL8 histopathology score: 0.0 ± 0.0 arbitrary units (AU)). Control subjects had completely preserved glomeruli and no IL8 staining (data not shown; and FIG. 3A ).

In addition, rt-PCR on the above biopsies showed that IL8 mRNA levels were upregulated in T2D patients compared with control individuals (IL8 mRNA: diabetic patients vs. controls: 3-fold increase, p < 0.05; Figure 3B). Briefly, RNA from purified glomeruli was extracted using Trizol reagent (Invitrogen) and qRTPCR analysis was performed using TaqMan analysis (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. Normalized expression values were determined using the ΔΔCt method. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) data were normalized to the expression of ACTB, and ΔΔCt values were calculated. Gene expression of all cell populations in each patient was compared by one-way ANOVA statistical analysis, followed by Bonferroni post hoc test for multiple comparisons of the target population and all other populations. Statistical analysis was also performed using the RT2 analyzer PCR array data analysis (Qiagen) available with software. No significant differences in CXCR1 and CXCR2 levels were observed, and detectable amounts of transcripts were identified (IL8 mRNA: diabetic vs. control, p < 0.05; Figure 7C). In addition, IL8 was expressed at the glomerular level and colocalized with synaptopodin (podocyte marker) and CD-31 (endothelial marker) (data not shown).

The data obtained revealed selective glomerular localization and co-expression with synaptopodin at the podocyte level and CD31 at the endothelial level (data not shown). Validating the evidence shown in Figures 3A and 3B, IL8 expression gradually decreased following the onset of renal parenchymal cellularity loss and fibrosis (data not shown).

Example 5

Urinary IL8 levels were higher in T2D patients with worsening renal function.

In order to confirm whether IL8 is associated with DKD in patients with type 2 diabetes (T2D), in particular, whether urine IL8 levels change according to albuminuria status, we used the Joslin group of individuals with T2D. The individuals included in this group suffer from T2D, followed by 8-12 years of follow-up, and examined renal function decline, proteinuria episodes, and end-stage renal disease (ESRD). The baseline level of urine IL8 was measured by Luminex in 1246 T2D patients, divided as follows: 702 normal albuminuria, 390 microalbuminuria, 156 macroalbuminuria, and 25 healthy subjects. In the first step, we evaluated the urine IL8 level at baseline and found that higher levels of urine IL8 levels in individuals with microalbuminuria were associated with the highest levels of albuminuria.

Briefly, use according to the manufacturer's protocol Milliplex based on magnetic microspheres Urine samples from each patient were tested for human IL8 concentration using the ELISA kit (EMD Millipore, Billerica MA) (4). Briefly, urine samples were allowed to thaw gradually at +4°C and then spun at 10,000 g for 10 minutes. The urine samples were then injected into magnetic beads and incubated overnight at +4°C with gentle shaking. The biotinylated reporter molecule was then added and the streptavidin-phycoerythrin solution was incubated with the sample at room temperature for 30 minutes. The samples were read using a Luminex Corp. Software package (Luminex Corp) analyzed the results. Data are expressed as mean ± standard error. When 2 groups were compared horizontally, bilateral unpaired Student's t test (for parametric data) or Mann-Whitney test (for non-parametric data) was used according to the distribution. When more than 2 groups were compared, ANOVA (for parametric data) and Kruskal-Wallis test (for non-parametric data) were used. P values less than 0.05 (by two-tailed test) were considered as indicators of statistical significance. Analyze data and create charts using GraphPad Prism software (GraphPad Software, Inc., SanDiego, CA).

The data obtained showed that patients with microalbuminuria showed higher urine IL8 levels compared with individuals with normal albuminuria [IL8: normal albuminuria = 19.69 ± 3.70 vs. microalbuminuria = 30.28 ± 5.42 pg / ml, p < 0.001; Figure 7A, Figure 7B and Figure 8A). Next, patients with microalbuminuria (more likely to develop impaired renal function) were evaluated, and the correlation between urine IL8 concentration and renal function loss was determined by measuring the urine albumin: creatinine ratio (ACR; mg / g). Patients with high urine IL8 at baseline (defined as better than the median distribution of IL8 in the microalbuminuria patient group) showed significantly worse renal function [ACR: Q3-Q4 (high IL8) = 101.7 ± 13.0 vs. Q1-Q2 (low IL8) = 58.5 ± 6.5 mg / g, p = 0.003, Figure 5B). The median threshold for IL8 is 2.41pg/ml. In addition, data from the Joslin group were analyzed; 389 patients with normal renal function (GFR≥60ml/min) and patients with albuminuria in the normal/micro range were followed up for 5 years. The albumin: creatinine ratio (ACR: mg/g) and GFR slope were calculated for all patients. Those patients with a distribution above the median of IL8 in the normal and microalbuminuria groups showed significantly higher ACR than those from patients below the median (Figure 8D). Among all 389 patients and their subsets of normal albuminuria and microalbuminuria, those patients who presented a positive test for IL8 in the urine also presented significantly higher ACR values (Figure 8B). The GFR slope during the 5-year follow-up was 1.6ml/year (IQR 0.5-3.3), and 55 patients had "hard kidney results" according to the FDA definition (ESRD, death from any cause or 30% loss of renal function). IL8 values were not quantitatively correlated with any of the follow-up variables considered (GFR slope, renal outcome risk, baseline proteinuria level). Subsequently, patients with a positive urine IL8 test were compared with patients with a negative urine IL8 test. Positive patients had a significantly greater GFR slope (2.47 ± 0.26 vs. 1.81 ± 0.16 ml/min/year p = 0.036, Figure 8C); This trend was also observed in the microalbuminuria group (2.5 ± 0.29 vs. 1.84 ± 0.19 ml/min/year p = 0.056, Figure 8C) and the normoalbuminuria group (2.3 ± 0.7 vs. 1.71 ± 0.28 ml/min/year p = 0.39, Figure 8C).

Regarding comprehensive renal outcomes, 368 of 389 patients had valid data; 41/55 events (74.5%) had positive urine IL8 values. The risk of events was 19% in IL8-positive patients, compared to 9% in IL8-negative patients, with an absolute risk increase of 10% and a relative increase of 111% (χ2 test, p=0.0096, data not shown). Among all subjects, those with IL8-positive urine had a risk of experiencing an event of 1.33 (95% C.I. 1.11 to 1.60) (ESRD, death from any cause or 30% loss of renal function). Similarly, the risks of early stages of microalbuminuria and normoalbuminuria were 1.29 and 1.76, respectively (95% C.I. 1.05 to 1.58 and 95% C.I. 1.41 to 2.20; Figure 8E).

Example 6

Identification of single nucleotide polymorphisms in the CXCR1 gene associated with DKD

In order to assess the importance of the IL8-CXR1/2 axis to human DN, samples from individuals with T2D from the Joslin nephropathy genetics study were assessed. Data from this group have been published (available at dbGaP, the World Wide Web page of the National Institutes of Health, accession number phs000302.v1.p1). Patients in this group were followed up for 8 to 12 years, and proteinuria, renal function decline and ESRD were examined. Experimental studies determine whether the accelerated progression of DN is associated with any spontaneous genetic variants of IL8, CXCR1 or CXCR2 loci. 326 T2D patients with or without DN were screened and genotyped to assess whether any single nucleotide polymorphisms (SNPs) at these loci can affect DN acceleration.

In detail, diabetic nephropathy cases from this collection were randomly selected for whole genome genotyping on the Illumina human CNV370v1 genotyping array (data available at dbGaP on the National Institutes of Health World Wide Web page, accession number phs000302.v1.p1) (Illumina, San Diego, CA). The application of quality control metrics of minimum allele frequency (MAF) < 0.01, rejection of the Hardy-Weinberg hypothesis (P ≤ 10-5) and difference rate of missing data (by case/control status) generated high-quality genotype data for 324,382 autosomal single nucleotide polymorphisms (SNPs). ACR and eGFR data from all individuals of European descent were used for quantitative trait analysis. P values were calculated using standard case/control allele tests. P values for quantitative trait analysis from estimated glomerular filtration rate are given. All association tests were performed using PLINK. SNP positions refer to NCBI Build 36.1 (3).

326 T2D individuals with and without DKD were screened and genotyped to assess whether any single nucleotide polymorphism (SNP) in the IL8, CXCR1, or CXCR2 genes would affect the onset of DKD. The strongest association with DKD in this population occurred at rs13006838 (log10-p value = 1.35), a SNP located at position 218461578 of the CXCR1 gene on chromosome 2.

Similarly, to confirm whether the IL8-CXCR1/2 axis is associated with diabetic kidney disease (DKD) in patients with T1D, we utilized the Genetics of Kidney Disease (GoKind) cohort (n = 829; 904 controls (2)). In this cohort, the strongest association with decreased estimated glomerular filtration rate (eGFR) occurred in the CXCR1 gene: (i) rs4674308 (chromosome 2, position 219018727), log10-p value = 1.36; (ii) rs4674309 (chromosome 2, position 219022817), log10-p value = 1.47; (iii) rs3755042 (chromosome 2, position 219025492), log10-p value = 1.36; (iv) rs7601872 (chromosome 2, position 219028129), log10-p value = 1.36; (v) rs664514 (chromosome 2, position 219038063), log10-p value = 1.31 (see Table 1 below).

SNPs at the IL8 and CXCR1/2 loci were observed to be associated with genotypes of DN, ACR, and eGFR. The strongest association with ESRD progression or GFR deterioration in the DN population occurred at rs13006838 (log10-p value = 1.35; p = 0.045); this SNP is located at position 218461578 of the CXCR1 gene on chromosome 2 (Table 4). This finding suggests the importance of the IL8-CXCR1/2 axis in the evolution of DN to ESRD. When GFR was adjusted for sex, age, body mass index, and glycated hemoglobin A1 C, this association was no longer significant. For CXCR2 and IL8, no loci associated with ESRD, ACR, or GFR progression were observed.

Example 7

IL8 and CXCR-1/2 are expressed by human podocytes

In vitro study of IL8, CXCR1 and CXCR2 expression of human podocytes (Figures 9A-9C). After complete cell differentiation in normal glucose (10mM [NG]) or high glucose (30mM [HG]), podocytes were cultured for 5 days. Mannitol (mannitol 20mM + glucose 10mM) was used as an osmotic control for high glucose. Immortalized podocyte lines were cultured using standard techniques known in the art. Weak expression of IL8 was detected under basal conditions, and it was not affected by glucose levels in the culture medium. In contrast, CXCR-1 decreased from 15% to less than 5% from normal to high blood sugar culture medium. No significant changes in CXCR-2 expression were detected (Figures 9B and 9C).

Unless otherwise stated, all in vitro and in vivo experimental data presented in the embodiments of this article were performed in triplicate. The proteinuria of db/db mice was carried out in 10 untreated and 30 treated animals. Data analysis was performed using Prism statistical software (LaJolla, CA-USA). Data were classified by D'Agostino-Pearsons test (continuous non-normally distributed variables were displayed by median and quartile range (IQR)), and analyzed using Mann-Whitney test (normally distributed variables were displayed by mean and standard deviation), and analyzed with two-tailed paired t test or one-way ANOVA. For discrete variables, the χ2 test was used. For all tests, p < 0.05 was considered significant.

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Table 1. CXCR1 sNPs associated with DN

Table 2. GFR levels in patients with progressively decreasing glomerular IL8 expression and progressive renal damage

Table 3. List of exemplary conditions identified for which a subject identified as suffering from one or a combination of the listed conditions can be treated using the methods disclosed herein

Claims (18)

1. A pharmaceutical composition for treating Diabetic Nephropathy (DN) or preventing or delaying the onset or progression of diabetic nephropathy in a subject who has been diagnosed with diabetes, comprising a CXCR1 and/or CXCR2 inhibitor.

2. The pharmaceutical composition of claim 1, wherein the CXCR1 and/or CXCR2 inhibitor is a compound selected from the group consisting of: r (-) -2- [ (4-isobutylphenyl) propionyl ] -methanesulfonamide, R (-) -2- [ (4' -trifluoromethanesulfonyl) oxy) phenyl ] -N-methanesulfonyl propionamide, and (2S) -2- (4- { [4- (trifluoromethyl) -1, 3-thiazol-2-yl ] amino } phenyl) propanoic acid and salts thereof.

3. The pharmaceutical composition of claim 1 or 2, wherein the CXCR1 and/or CXCR2 inhibitor is a compound selected from the group consisting of: lysine salt of R (-) -2- [ (4-isobutylphenyl) propionyl ] -methanesulfonamide, sodium salt of R (-) -2- [ (4' -trifluoromethanesulfonyl) phenyl ] -N-methanesulfonyl propionamide, and sodium salt of (2S) -2- (4- { [4- (trifluoromethyl) -1, 3-thiazol-2-yl ] amino } phenyl).

4. The pharmaceutical composition of any one of claims 1-3, wherein the diabetes is type 1 diabetes (T1D) or type 2 diabetes (T2D).

5. The pharmaceutical composition of any one of claims 1-4, wherein the subject has at least one of the following single nucleotide polymorphisms at the CXCR1 locus: s13006838, rs4674308; rs4674309; rs3755042; rs7601872; and rs664514.

6. The pharmaceutical composition of any one of claims 1-5, further comprising measuring protein levels in a urine sample from the subject.

7. The pharmaceutical composition of claim 6, wherein the subject has been determined to have an albumin excretion rate of 30mg to 300mg per day.

8. The pharmaceutical composition of claim 6, further comprising comparing the measured urine protein level to a urine protein reference, wherein the urine protein reference is a protein level in a urine sample obtained in a normal healthy subject not suffering from any kidney disease.

9. The pharmaceutical composition of any one of claims 1-8, further comprising measuring the level of IL8 in a sample obtained from the subject.

10. The pharmaceutical composition of claim 9, wherein the subject has been determined to have a urinary IL8 level of greater than 2.41 pg/ml.

11. The pharmaceutical composition according to claim 9 or 10, wherein the sample is a urine sample, a kidney biopsy sample, a serum sample, a blood sample or a plasma sample.

12. The pharmaceutical composition of claim 10 or 11, further comprising comparing the measured IL8 level to an IL8 reference, wherein the IL8 reference is the IL8 level in a corresponding sample obtained in a normal healthy subject not suffering from any kidney disease.

13. The pharmaceutical composition of any one of claims 1-12, wherein the CXCR1 and/or CXCR2 inhibitor is administered to the subject prior to, concurrently with, or after administration of at least one therapy for diabetes, metabolic syndrome, cardiovascular disease, and hypertension.

14. The pharmaceutical composition of any one of claims 1-13, wherein the CXCR1 and/or CXCR2 inhibitor is administered together with at least one active molecule for the treatment of diabetes.

15. The pharmaceutical composition of any one of claims 1-14, wherein the CXCR1 and/or CXCR2 inhibitor is in a composition formulated for delivery to the kidney.

16. The pharmaceutical composition of any one of claims 1-15, wherein the CXCR1 and/or CXCR2 inhibitor is administered by a systemic route, an enteral route, or a parenteral route.

17. The pharmaceutical composition of any one of claims 1-16, wherein the daily dose of CXCR1 and/or CXCR2 inhibitor is 1mg-100mg.

18. The pharmaceutical composition of any one of claims 1-17, wherein the subject has been determined to have a volume of 60ml/min/1.73m ² The Glomerular Filtration Rate (GFR) values above.